Selectin inhibitors, composition, and uses related thereto

ABSTRACT

This disclosure relates to selectin inhibitors, compositions, and methods related thereto. In certain embodiments, the disclosure relates to glycopeptides that contain one or more modified amino acids conjugated to a saccharide or polysaccharide. In certain embodiments, the disclosure relates to uses of the glycopeptides as anti-inflammatory, antithrombotic, or anti-metastatic agents.

CROSS REFERENCE TO RELATED APPLICATIONS

This Application claims priority to U.S. Provisional Application No.61/830,285 filed Jun. 3, 2013, hereby incorporated by reference in itsentirety.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

This invention was made with government support under Grants DK069275,HL106018, HL60963, and HL085607 awarded by the National Institutes ofHealth. The government has certain rights in the invention.

BACKGROUND

The selectin family of cell adhesion molecules, together with theirglycoconjugate ligands, participate in leukocyte trafficking to sites ofinflammation and to lymphoid organs. P- and E-selectins are expressed onactivated vascular endothelial cells where they mediate initialtethering and rolling of leukocytes on endothelial cells by binding toP-selectin glycopeptide ligand-1 (PSGL-1) present on the surface ofleukocytes. P-selectin is also expressed on activated platelets.L-selectin is expressed on the surface of leukocytes and mediatesleukocyte-leukocyte interactions by binding to PSGL-1 present on thesurface of other leukocytes promoting leukocyte accumulation to theinflammatory sites. Selectins recognize the sialyl Lewis x epitope(SLex, NeuAcα2-3Galβ1-4(Fucα1-3)GlcNAcβ1-) on glycoconjugate ligands.However, selectin binding to SLex determinant alone is low affinity andis necessary but not sufficient for physiological interactions. Thus,selectins require additional post-translational modifications or peptidecomponents for high-affinity binding to their ligands. P- and L-selectinboth bind to the extreme N-terminus of PSGL-1 and interact with threeclustered tyrosine sulfate residues and a nearby core-2-based O-glycanwith sialyl Lewis x epitope (C2-SLex). The N-terminus of human PSGL-1contains three potential tyrosine sulfation sites (Y46, Y48 and Y51) andtwo potential O-glycan attachment sites (T44 and T57).

Leppanen et al. report that binding of glycosulfopeptides to P-selectinimplicates stereospecific contributions of individual tyrosine sulfateand sugar residues. J Biol Chem., 2000, 275(50):39569-39578. Leppanen etal. also report that human L-selectin preferentially binds syntheticglycosulfopeptides modeled after endoglycan. Glycobiology, 2010,20(9):1170-1185. See also WO 2003/032925, WO 99/65712, US 2002/0026033,U.S. Pat. Nos. 5,858,994, 6,136,790, Hicks et al. FASEB J, 2002,16(11):1461-1462, and Hicks et al. FASEB J, 2002, 16(5):A1052.

Ohta et al. report inhibition of P-selectin specific cell adhesion by alow molecular weight, non-carbohydrate compound, KF38789. Inflamm Res,2001, 50(11):544-51.

Several small molecule inhibitors and protein therapeutics aimed atblocking PSGL-1/P-selectin interactions are already in clinical trials.Certain candidates pose production, stability, and immunity issues.Thus, there is a need for molecules that bind to selectins with highspecificity and affinity, which in turn inhibit selectin mediatedcell-cell interactions that have desirable pharmacological properties.

References cited herein are not an admission of prior art.

SUMMARY

This disclosure relates to selectin inhibitors, compositions, andmethods related thereto. In certain embodiments, the disclosure relatesto glycopeptides that contain one or more modified amino acidsconjugated to a saccharide or polysaccharide. In certain embodiments,the disclosure relates to uses of the glycopeptides asanti-inflammatory, antithrombotic, or anti-metastatic agents.

In certain embodiments, the disclosure relates to isolated non-naturallyoccurring glycopeptides comprising Y¹X¹Y²X²X³Y³X⁴X⁵X⁶Z¹X⁷W¹ (SEQ IDNO: 1) or salts thereof, wherein

W¹ is threonine or serine substituted with a saccharide orpolysaccharide,

X¹, X², X³, X⁴, X⁵, X⁶, and X⁷ individually and independently any aminoacid,

Y¹, Y², and Y³ are each individually and independently tyrosine,phenylalanine, or phenylglycine unsubstituted or substituted with —SO₃H,—CH₂SO₃H, —CF₂SO₃H, —CO₂H, —CONH₂, —NHSO₂CH₃, —SO₂NH₂, or —CH₂PO₃H, and

Z¹ is proline or hydroxyproline.

In certain embodiment, the disclosure relates to pharmaceuticalcompositions comprising a glycopeptides disclosed herein and apharmaceutically acceptable excipient. In certain embodiments, thepharmaceutical formulation is in the form of a pill, tablet, capsule, orgel or in the form of an aqueous saline buffer wherein thepharmaceutically acceptable excipient is a saccharide or polysaccharide.

In certain embodiments, this disclosure relates to methods of treatingor preventing vascular disease or conditions such as atherosclerosis,atherosclerotic lesions, thrombus formation, thromboembolism, stroke, ormyocardial infarction by administering an effective amount of apharmaceutical composition disclosed herein to a subject in needthereof. In certain embodiments, the subject is at risk of, exhibitingsymptoms of, or diagnosed with atherosclerosis, atherosclerotic lesions,thrombus formation, thromboembolism, stroke, or myocardial infarction.

In certain embodiments, the disclosure contemplates methods of treatingor preventing allergies or lung diseases or conditions comprisingadministering an effective amount of a pharmaceutical compositiondisclosed herein to a subject in need thereof. In certain embodiments,the subject is at risk of, exhibiting symptoms of, or diagnosed withasthma, bronchitis, emphysema, and COPD.

In certain embodiments, the disclosure relates to methods of treating orpreventing cancer or tumor metastasis comprising administering aneffective amount of a pharmaceutical composition disclosed herein to asubject in need thereof.

In certain embodiments, the disclosure relates to the production of amedicament comprising glycosulfopeptides disclosed herein for usesdisclosed herein.

In certain embodiments, the disclosure relates to methods of producingpolysaccharides and glycopeptides disclosed herein comprising mixingstarting materials and reagents under conditions such that the productsare formed.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates certain glycosulfopeptides mimetics of PSGL-1.

FIG. 2 illustrates the synthesis of the Core-2 glycan and subsequentenzymatic steps to afford a family of glycopeptide mimetics of PSGL-1.Enzymatic steps (a) UDP-Gal, β-1,4-GalT (bovine), alkaline phosphatase,130 mM HEPES, pH 7.4, 40 mM sodium cacodylate, pH 7.0, 20 mM MnCl2, and0.02% NaN3; (b) α 2,3-(N)-sialylT CMP-NeuAc 50 mM MOPS, pH 7.4, 0.1%bovine serum albumin, and 0.02% NaN3, 14 h; (c) GDP-Fuc, α-1,3-FucT-VI,50 mM MOPS, pH 7.4, 20 mM MnCl₂ and 0.02% NaN3, 16 h. Disialyl GSnP-6was obtained from GSnP-4 in 45% yield. GSnP-7 was obtained in 55% yieldby fucosylation of disialyl GSnP-6. 4-(Sulfomethyl) series (n): GSnP-6(X: CH2; R=H); GSnP-7 (X: CH2; R=Sialyl); 4-(Sulfo)phenylalanine series(n2): GSn2P-6 (X: bond; R=H); Tyrosine O-sulfate series: GSP-6 (X: O;R=H). The last numeral refers to the size of the glycan (e.g. 6 forhexasaccharide).

FIG. 3 shows data on microarray binding screen of glycopeptide mimics to(A) human and mouse P-selectin (5 μg/mL), (B) human and mouse L-selectin(20 μg/mL), and (C) human and mouse E-selectin (20 μg/mL) Referencecompounds included Sialyl Lewis x (SLe^(x)), the biantennary glycansNA2, NA2,3, NA2,6, mannotriose-di-(N-acetyl-D-glucosamine), as well aslacto-N-neo-tetraose (LNnT) and biotin. Bound peptides were detectedusing Alexa-488 labeled anti-human IgG antibody (5 μg/mL). Three lectinsRCA-1, AAL, and PNA were used to confirm the sequence of enzymaticsteps. Monoclonal antibodies CHO131, PSG2 antibody and PL-1 were used toconfirm the presence of SLex, tyrosine sulfates, and the peptidesequence, respectively. Biacore binding analysis to human P-selectinwith observed rate constants for (D) GSnP-6, k_(on) 3.1×10⁵ M⁻¹s⁻¹,k_(off) 6.9×10³ M⁻¹s⁻; GSn₂P-6, k_(on) 6.4×10⁵ M⁻¹s⁻¹, k_(off) 6.8×10³M⁻¹s. Biacore binding analysis to mouse P-selectin with observed rateconstants for (E) GsnP-6, k_(on) 4.9×10⁴ M⁻¹s⁻¹, k_(off) 8.0×10³ M⁻¹s⁻;GSn₂P-6 k_(on) 5.3×10⁴ M⁻¹s⁻¹, k_(off) 9.0×10³ M⁻¹s. (F) Temperature-and pH-dependent stability studies of GSnP-6.

FIG. 4 shows data on (A) Florescence activated cell sorting (FACS)performed to determine the capacity of GSnP-6 to inhibit P andL-selectin binding to murine and U937 leukocytes, as well as to (B)human peripheral blood monocytes and neutrophils. (C) Intravitalmicroscopy of the murine cremaster muscle microcirculation demonstratedincreased leukocyte rolling velocity after intravenous administration ofGSnP-6 (4 μmol/kg IV; p≤0.01).

DETAILED DESCRIPTION

Before the present disclosure is described in greater detail, it is tobe understood that this disclosure is not limited to particularembodiments described, and as such may, of course, vary. It is also tobe understood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present disclosure will be limited onlyby the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present disclosure, the preferredmethods and materials are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present disclosure is not entitled to antedate suchpublication by virtue of prior disclosure. Further, the dates ofpublication provided could be different from the actual publicationdates that may need to be independently confirmed.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentdisclosure. Any recited method can be carried out in the order of eventsrecited or in any other order that is logically possible.

Embodiments of the present disclosure will employ, unless otherwiseindicated, techniques of medicine, organic chemistry, biochemistry,molecular biology, pharmacology, physiology, and the like, which arewithin the skill of the art. Such techniques are explained fully in theliterature.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise. In this specification andin the claims that follow, reference will be made to a number of termsthat shall be defined to have the following meanings unless a contraryintention is apparent.

Prior to describing the various embodiments, the following definitionsare provided and should be used unless otherwise indicated.

As used herein, “salts” refer to derivatives of the disclosed compoundswhere the parent compound is modified making acid or base salts thereof.Examples of salts include, but are not limited to, mineral or organicacid salts of basic residues such as amines, alkylamines, ordialkylamines; alkali or organic salts of acidic residues such ascarboxylic acids e.g., sodium or potassium salts of sulfonic acid, andthe like. In typical embodiments, the salts are conventional nontoxicpharmaceutically acceptable salts including the quaternary ammoniumsalts of the parent compound formed, and non-toxic inorganic or organicacids. Preferred salts include those derived from inorganic acids suchas hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric andthe like; and the salts prepared from organic acids such as acetic,propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric,ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic,benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric,toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic,and the like.

“Subject” refers any animal, preferably a human patient, livestock,rodent, monkey or domestic pet.

As used herein, the terms “prevent” and “preventing” include theprevention of the recurrence, spread or onset. It is not intended thatthe present disclosure be limited to complete prevention. In someembodiments, the onset is delayed, or the severity of the disease isreduced.

As used herein, the terms “treat” and “treating” are not limited to thecase where the subject (e.g., patient) is cured and the disease iseradicated. Rather, embodiments, of the present disclosure alsocontemplate treatment that merely reduces symptoms, and/or delaysdisease progression.

As used herein, the term “combination with” when used to describeadministration with an additional treatment means that the agent may beadministered prior to, together with, or after the additional treatment,or a combination thereof.

A “protecting group” refers to those moieties that are introduced into amolecule by chemical modification of a functional group in order toobtain chemoselectivity in a subsequent chemical reaction or tofacilitate purification. Protecting groups may be categorized by thereaction conditions and/or reagents that are used to remove them such asacid labile protecting groups, base labile protecting groups andhydrogenation removable protecting groups. For example, acid labileprotecting groups, such as tBu or Boc, typically decompose when exposedto strong acidic conditions providing a hydrogen substituent in place oftBu or Boc protecting group. Acetyl esters and thioesters of alcoholsand thiols are examples of base labile protecting groups. Additionalexamples of protecting groups include, but are not limited to,4-methoxy-2,3,6-trimethylphenyl)sulfonyl (Mtr),2,2,5,7,8-pentamethyl-chroman-6-sulphonyl (Pmc), tosyl (Tos),mesitylenesulfonyl (Mts), 4,4′-dimethoxybenzhydryl (Mbh),2,4,6-trimethoxybenzyl (Tmob), tripheylmethyl (Trt),9-fluorenylmethyloxycarbonyl (fmoc), tert-butyl (tBu), benzyl (Bzl),t-butoxymethyl ether (Bum), (2,4-dinitrophenol) Dnp, benzyloxymethyl(Bom), benzyloxycarbonyl (Z), 2-chloro-benzyloxycarbonyl (CIZ),t-butyloxycarbonyl (Boc), formyl (CHO) or 2-bromobenzyloxycarbonyl (BrZ)and heterocycles such as succinimide, maleimide, and phathalimide.Protecting groups may be in the form of derivatives, e.g., having one ormore substituents.

As used herein, the term “derivative” refers to a structurally similarcompound that retains sufficient functional attributes of the identifiedanalogue. The derivative may be structurally similar because it islacking one or more atoms, substituted, a salt, in differenthydration/oxidation states, or because one or more atoms within themolecule are switched, such as, but not limited to, replacing an oxygenatom with a sulfur or nitrogen atom or replacing an amino group with ahydroxyl group or vice versa. The derivative may be a prodrug.Derivatives may be prepare by any variety of synthetic methods orappropriate adaptations presented in synthetic or organic chemistry textbooks, such as those provide in March's Advanced Organic Chemistry:Reactions, Mechanisms, and Structure, Wiley, 6th Edition (2007) MichaelB. Smith or Domino Reactions in Organic Synthesis, Wiley (2006) Lutz F.Tietze hereby incorporated by reference.

The term “substituted” refers to a molecule wherein at least onehydrogen atom is replaced with a substituent. When substituted, one ormore of the groups are “substituents.” The molecule may be multiplysubstituted. In the case of an oxo substituent (“═O”), two hydrogenatoms are replaced. Example substituents within this context may includehalogen, hydroxy, alkyl, alkoxy, nitro, cyano, oxo, carbocyclyl,carbocycloalkyl, heterocarbocyclyl, heterocarbocycloalkyl, aryl,arylalkyl, heteroaryl, heteroarylalkyl, —NR_(a)R_(b), —NR_(a)C(═O)R_(b),—NR_(a)C(═O)NR_(a)NR_(b), —NR_(a)C(═O)OR_(b), —NR_(a)SO₂R_(b),—C(═O)R_(a), —C(═O)OR_(a), —C(═O)NR_(a)R_(b), —OC(═O)NR_(a)R_(b),—OR_(a), —SR_(a), —SOR_(a), —S(═O)₂R_(a), —OS(═O)₂R_(a) and—S(═O)₂OR_(a). R_(a) and R_(b) in this context may be the same ordifferent and independently hydrogen, halogen hydroxyl, alkyl, alkoxy,alkyl, amino, alkylamino, dialkylamino, carbocyclyl, carbocycloalkyl,heterocarbocyclyl, heterocarbocycloalkyl, aryl, arylalkyl, heteroaryl,heteroarylalkyl.

Substituted Glycosulfopeptides Derivatives

A tyrosine derivative, 4-sulfonomethyl phenylalanine, chemical name2-amino-3-(4-(sulfonomethyl)phenyl)propanoic acid, was incorporated intoglycosulfopeptides as illustrated in FIG. 1. A chemoenzymatic syntheticscheme was developed for the generation of selectin ligandglycosulfopeptides. The amino acid Phe (p-CH₂SO₃H) may be preparedaccording to the procedures in Roosenburg et al., Bioconjugate Chem,2010, 21, 663-670. Fmoc-Phe(4-CH₂—SO₃H) and other amino acids, such asFmoc-Phg(4-CH₂—SO₃Na), Fmoc-Phe(4-NO₂), Fmoc-Phe(4-COO-tBu),Fmoc-Phe(4-C(O)NH₂), Fmoc-Phe(4-NH—SO₂—CH₃), andBoc-L-Phe[4-CH₂—PO₃(bn)₂]—OMe, are commercially available from RSP aminoacids LLC, Shirley, Mass.

Certain embodiments were identified with nanomolar binding affinity toP-selectin. Blockade of P-selectin/PSGL-1 interactions inhibit leukocyterecruitment during inflammation. Certain substituted glycosulfopeptidesderivatives modeled after N-terminus PSG1-1 sequence are P-selectinantagonist that are uniquely stable during chemical synthesis, and theyare suitable for preparative scale synthesis. PSGL-1 normally containstyrosine sulfate moieties which are typically unstable during chemicalsynthesis. Since sulfonate isosters are resistant to both hydrolysis andoxidation, the replacement of the tyrosine sulfate (X=OSO₃) moiety witha robust isosteric C-sulfonate (X=CH₂SO₃) overcomes the stabilityproblem and affords compounds with high levels of P-selectin affinity.In the sulfonate analog (X=SO₃), although missing the oxygen atomlinking the SO₃ and phenyl group, it retains the aromatic and anionicproperties of tyrosine sulfate, and thus serves as a stable mimetic.

In certain embodiments, the disclosure relates to isolated glycopeptidescomprising Y¹X¹Y²X²X³Y³X⁴X⁵X⁶Z¹X⁷W¹ (SEQ ID NO: 1) or salts thereof,wherein

W¹ is threonine or serine substituted with a saccharide orpolysaccharide,

X¹, X², X³, X⁴, X⁵, X⁶, and X⁷ individually and independently any aminoacid,

Y¹, Y², and Y³ are each individually and independently tyrosine,phenylalanine, or phenylglycine unsubstituted or substituted with —SO₃H,—CH₂SO₃H, —CO₂H, —CONH₂, —NHSO₂CH₃, or —CH₂PO₃H, and

Z¹ is proline or hydroxyproline.

In certain embodiments, at least one or two of Y¹, Y², and Y³ arephenylalanine 4-substituted with sulfonomethyl.

In certain embodiments, all Y¹, Y², and Y³ are phenylalanine4-substituted with sulfonomethyl.

In certain embodiments, the polysaccharide is sialyl Lewis X or sialylLewis A. In certain embodiments, the polysaccharide comprises,

2-(acetylamino)-2-deoxy-galactose alpha 1 bonded to W¹,

galactose beta 3 bonded to 2-(acetylamino)-2-deoxy-galactose,

2-(acetylamino)-2-deoxy-glucose beta 6 bonded to2-(acetylamino)-2-deoxy-galactose and

Fucose is alpha 3 bonded to 2-(acetylamino)-2-deoxy-glucose.

In certain embodiments, the polysaccharide comprises,

2-(acetylamino)-2-deoxy-galactose alpha 1 bonded to W¹,

galactose beta 3 bonded to 2-(acetylamino)-2-deoxy-galactose,

2-(acetylamino)-2-deoxy-glucose beta 6 bonded to2-(acetylamino)-2-deoxy-galactose,

fucose is alpha 3 bonded to 2-(acetylamino)-2-deoxy-glucose, and

galactose beta 4 bonded to 2-(acetylamino)-2-deoxy-glucose.

In certain embodiments, the polysaccharide comprises,

2-(acetylamino)-2-deoxy-galactose alpha 1 bonded to W¹,

galactose beta 3 bonded to 2-(acetylamino)-2-deoxy-galactose,

2-(acetylamino)-2-deoxy-glucose beta 6 bonded to2-(acetylamino)-2-deoxy-galactose,

fucose is alpha 3 bonded to 2-(acetylamino)-2-deoxy-glucose,

galactose beta 4 bonded to 2-(acetylamino)-2-deoxy-glucose,

5-acetamido-3,5-dideoxy-glycero-galacto-2-nonulosonic acid alpha 3bonded to galactose.

In certain embodiments, the polysaccharide comprises,

2-(acetylamino)-2-deoxy-galactose alpha 1 bonded to W¹,

galactose beta 3 bonded to 2-(acetylamino)-2-deoxy-galactose,

2-(acetylamino)-2-deoxy-glucose beta 6 bonded to2-(acetylamino)-2-deoxy-galactose,

galactose beta 4 bonded to 2-(acetylamino)-2-deoxy-glucose,

5-acetamido-3,5-dideoxy-glycero-galacto-2-nonulosonic acid alpha 3bonded to galactose, and

fucose is alpha 3 bonded to 2-(acetylamino)-2-deoxy-glucose.

In certain embodiments, the polysaccharide comprises,

2-(acetylamino)-2-deoxy-galactose alpha 1 bonded to W¹,

a first galactose beta 3 bonded to 2-(acetylamino)-2-deoxy-galactose,

2-(acetylamino)-2-deoxy-glucose beta 6 bonded to2-(acetylamino)-2-deoxy-galactose,

a second galactose beta 4 bonded to 2-(acetylamino)-2-deoxy-glucose,

5-acetamido-3,5-dideoxy-glycero-galacto-2-nonulosonic acid is alpha 3bonded to the second galactose,

fucose is alpha 3 bonded to 2-(acetylamino)-2-deoxy-glucose, and

5-acetamido-3,5-dideoxy-glycero-galacto-2-nonulosonic acid is alpha 3bonded to the first galactose.

In certain embodiments, X¹, X³, X⁴, and X⁷ is each individually andindependently E, D, N, or Q.

In certain embodiments, X², X⁵, and X⁶ is each individually andindependently L, I, V, A or F.

In certain embodiments, in certain embodiments, the disclosure relatesto isolated glycopeptides comprising or consisting of

(SEQ ID NO: 2) Y¹EY²LDY³DFLZ¹EW¹E, (SEQ ID NO: 3) Y¹EY²LDY³DFLZ¹EW¹EP,(SEQ ID NO: 4) Y¹EY²LDY³DFLZ¹EW¹EPL, (SEQ ID NO: 5) EY¹EY²LDY³DFLZ¹EW¹,(SEQ ID NO: 6) EY¹EY²LDY³DFLZ¹EW¹E, (SEQ ID NO: 7) EY¹EY²LDY³DFLZ¹EW¹EP,(SEQ ID NO: 8) EY¹EY²LDY³DFLZ¹EW¹EPL, (SEQ ID NO: 9)KEY¹EY²LDY³DFLZ¹EW¹, (SEQ ID NO: 10) KEY¹EY²LDY³DFLZ¹EW¹E,(SEQ ID NO: 11) KEY¹EY²LDY³DFLZ¹EW¹EP, or (SEQ ID NO: 12)KEY¹EY²LDY³DFLZ¹EW¹EPL.

In certain embodiments, the disclosure relates to an isolatedglycopeptides comprising KEY¹EY²LDY³DFLZ¹EW¹EPL (SEQ ID NO: 12) or saltthereof wherein,

W¹ is threonine,

Y¹, Y², and Y³ are phenylalanine 4-substituted with sulfonomethyl,

Z¹ is proline;

2-(acetylamino)-2-deoxy-galactose alpha 1 bonded to W¹,

galactose beta 3 bonded to 2-(acetylamino)-2-deoxy-galactose,

2-(acetylamino)-2-deoxy-glucose beta 6 bonded to2-(acetylamino)-2-deoxy-galactose,

fucose is alpha 3 bonded to 2-(acetylamino)-2-deoxy-glucose,

galactose beta 4 bonded to 2-(acetylamino)-2-deoxy-glucose, and

5-acetamido-3,5-dideoxy-glycero-galacto-2-nonulosonic acid is alpha 3bonded to galactose.

In certain embodiments, the disclosure relates to compositionscomprising intermediate compounds such as of the following formula,

wherein R1 is hydrogen, a protecting group, an amino protecting group,or a base removable protecting group and

R2 is hydrogen, a protecting group, a carboxylic acid protecting groupor a acid removable protecting group or

wherein R1 is hydrogen, a carboxylic acid protecting group or a acidremovable protecting group and

R2 is hydrogen, amino protecting group or a base removable protectinggroup and

R3 is hydrogen, an amino acid side chain, or alkyl optionallysubstituted with one or more substituent.

In certain embodiments, the base removable protecting group is theheterocyclic base removable protecting group Fmoc.

In certain embodiments, the acid removable protecting group istert-butyl or Boc.

Pharmaceutical Compositions

In certain embodiment, the disclosure relates to pharmaceuticalcompositions comprising a glycopeptides disclosed herein and apharmaceutically acceptable excipient. In certain embodiments, thepharmaceutical formulation is in the form of a pill, tablet, capsule, orgel or in the form of an aqueous saline buffer wherein thepharmaceutically acceptable excipient is a saccharide or polysaccharide.

As used herein the language “pharmaceutically acceptable excipient” isintended to include any and all carriers, solvents, diluents,excipients, adjuvants, dispersion media, coatings, antibacterial andantifungal agents, and absorption delaying agents, and the like,compatible with pharmaceutical administration.

Suitably, the pharmaceutical composition of the disclosure comprises acarrier and/or diluent appropriate for its delivering by injection to ahuman or animal organism. Such carrier and/or diluent is non-toxic atthe dosage and concentration employed. It is selected from those usuallyemployed to formulate compositions for parental administration in eitherunit dosage or multi-dose form or for direct infusion by continuous orperiodic infusion. It is typically isotonic, hypotonic or weaklyhypertonic and has a relatively low ionic strength, such as provided bysugars, polyalcohols and isotonic saline solutions. Representativeexamples include sterile water, physiological saline (e.g. sodiumchloride), bacteriostatic water, Ringer's solution, glucose orsaccharose solutions, Hank's solution, and other aqueous physiologicallybalanced salt solutions (see for example the most current edition ofRemington: The Science and Practice of Pharmacy, A. Gennaro, Lippincott,Williams & Wilkins). The pH of the composition of the disclosure issuitably adjusted and buffered in order to be appropriate for use inhumans or animals, typically at a physiological or slightly basic pH(between about pH 8 to about pH 9, with a special preference for pH8.5). Suitable buffers include phosphate buffer (e.g. PBS), bicarbonatebuffer and/or Tris buffer. A typical composition is formulated in 1Msaccharose, 150 mM NaCl, 1 mM MgCl2, 54 mg/l Tween 80, 10 mM Tris pH8.5. Another typical composition is formulated in 10 mg/ml mannitol, 1mg/ml HSA, 20 mM Tris, pH 7.2, and 150 mM NaCl.

The composition of the disclosure can be in various forms, e.g. in solid(e.g. powder, lyophilized form), or liquid (e.g. aqueous). In the caseof solid compositions, the typical methods of preparation are vacuumdrying and freeze-drying which yields a powder of the active agent plusany additional desired ingredient from a previously sterile-filteredsolution thereof. Such solutions can, if desired, be stored in a sterileampoule ready for reconstitution by the addition of sterile water forready injection.

Nebulized or aerosolized formulations also form part of this disclosure.Methods of intranasal administration include the administration of adroplet, spray, or dry powdered form of the composition into thenasopharynx of the individual to be treated from a pressured containeror dispenser which contains a suitable propellant, e.g., a gas such ascarbon dioxide, or a nebulizer (see for example WO 95/11664). Entericformulations such as gastroresistant capsules and granules for oraladministration, suppositories for rectal or vaginal administration alsoform part of this disclosure. For non-parental administration, thecompositions can also include absorption enhancers which increase thepore size of the mucosal membrane. Such absorption enhancers includesodium deoxycholate, sodium glycocholate, dimethyl-beta-cyclodextrin,lauroyl-1-lysophosphatidylcholine and other substances having structuralsimilarities to the phospholipid domains of the mucosal membrane.

The composition can also contain other pharmaceutically acceptableexcipients for providing desirable pharmaceutical or pharmacodynamicproperties, including for example modifying or maintaining the pH,osmolarity, viscosity, clarity, color, sterility, stability, rate ofdissolution of the formulation, modifying or maintaining release orabsorption into an the human or animal organism. For example, polymerssuch as polyethylene glycol may be used to obtain desirable propertiesof solubility, stability, half-life and other pharmaceuticallyadvantageous properties (Davis et al., 1978, Enzyme Eng. 4, 169-173;Burnham et al., 1994, Am. J. Hosp. Pharm. 51, 210-218). Representativeexamples of stabilizing components include polysorbate 80, L-arginine,polyvinylpyrrolidone, trehalose, and combinations thereof. Viscosityenhancing agents include sodium carboxymethylcellulose, sorbitol, anddextran. The composition can also contain substances known in the art topromote penetration or transport across the blood barrier or membrane ofa particular organ (e.g. antibody to transferrin receptor; Friden etal., 1993, Science 259, 373-377). A gel complex of poly-lysine andlactose (Midoux et al., 1993, Nucleic Acid Res. 21, 871-878) orpoloxamer 407 (Pastore, 1994, Circulation 90, 1-517) can be used tofacilitate administration in arterial cells.

The composition may be administered to patients in an amount effective,especially to enhance an immune response in an animal or human organism.As used herein, the term “effective amount” refers to an amountsufficient to realize a desired biological effect. The appropriatedosage may vary depending upon known factors such as the pharmacodynamiccharacteristics of the particular active agent, age, health, and weightof the host organism; the condition(s) to be treated, nature and extentof symptoms, kind of concurrent treatment, frequency of treatment, theneed for prevention or therapy and/or the effect desired. The dosagewill also be calculated dependent upon the particular route ofadministration selected. Further refinement of the calculationsnecessary to determine the appropriate dosage for treatment is routinelymade by a practitioner, in the light of the relevant circumstances. Thetiter may be determined by conventional techniques. The administrationmay take place in a single dose or a dose repeated one or several timesafter a certain time interval.

Methods of Use

P-selectin plays an important role during the initial steps of leukocyterecruitment to the sites of inflammation. Thus, the development oftherapeutics that block P-selectin/PSGL-1 interactions is desirable.Substituted tyrosine glycosulfopeptides derivatives such as GSnP-6 couldbe useful for treating atherosclerosis, inflammatory bowel disease,sickle cell disease, ischemia and reperfusion injury, coagulopathies,lung diseases, tumor metastasis, as well as having diagnosticapplications.

Selectins promote tumor metastasis. See Laubli & Borsig, Semin CancerBiol, 2010, 20(3):169-77. Gong et al. report P-selectin-mediatedplatelet activation promotes adhesion of non-small cell lung carcinomacells on vascular endothelial cells. Mol Med Rep, 2012, 5(4):935-42. Cuiet al. report differential expression of the alpha 2,3-sialic acidresidues in breast cancer is associated with metastatic potential. OncolRep, 2011, 25(5):1365-71. See also Shirure et al., PLoS One. 2012,7(9):e44529 and Perez-Garay et al., PLoS One, 2010, 5(9):e12524.

In certain embodiments, the disclosure relates to methods of treating orpreventing cancer or tumor metastasis comprising administering aneffective amount of a pharmaceutical composition disclosed herein to asubject in need thereof.

In certain embodiments, the disclosure relates to methods of treating orpreventing cancer comprising administering an effective amount of acompound disclosed herein to a subject in need thereof. In certainembodiments, the subject diagnosed with, exhibiting symptoms of, or atrisk of cancer. In certain embodiments, the cancer is venous ulcers,angiogenic disorders of the skin, a hematological malignancy, aleukemia, lymphoma, acute lymphoblastic leukemia (ALL), acutemyelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), smalllymphocytic lymphoma (SLL), chronic myelogenous leukemia, acutemonocytic leukemia (AMOL), Hodgkin's lymphomas, non-Hodgkin's lymphomas,Burkitt lymphoma, B-cell lymphoma, multiple myelomacervical, ovariancancer, colon cancer, breast cancer, gastric cancer, lung cancer,melanoma, skin cancer, ovarian cancer, pancreatic cancer, prostatecancer, head cancer, neck cancer, and renal cancer.

In certain embodiments, cancer therapeutic strategies entailpharmaceutical compositions comprising a glycopeptide disclosed hereinadministered in combination with a second anti-cancer agent such asgefitinib, erlotinib, docetaxel, cis-platin, 5-fluorouracil,gemcitabine, tegafur, raltitrexed, methotrexate, cytosine arabinoside,hydroxyurea, adriamycin, bleomycin, doxorubicin, daunomycin, epirubicin,idarubicin, mitomycin-C, dactinomycin and mithramycin, vincristine,vinblastine, vindesine, vinorelbine taxol, taxotere, etoposide,teniposide, amsacrine, topotecan, camptothecin, bortezomib, anagrelide,tamoxifen, toremifene, raloxifene, droloxifene, iodoxyfene, fulvestrant,bicalutamide, flutamide, nilutamide, cyproterone, goserelin,leuprorelin, buserelin, megestrol, anastrozole, letrozole, vorazole,exemestane, finasteride, marimastat, trastuzumab, cetuximab, dasatinib,imatinib, bevacizumab, combretastatin, thalidomide, and/or lenalidomideor combinations thereof.

Certain selectins activate cell adhesion to the vascular endotheliumallowing them to infiltrate tissue from the circulating blood. P-, E-,and L-selectin mediate migration of activated CD8⁺ T lymphocytes intoinflamed skin. Hirata et al., J Immunol, 2002, 169:4307-13. Culmer etal. report that circulating and vein wall P-selectin promote venousthrombogenesis during aging in a rodent model and assert that this isevidence supporting the use of selectin targeted therapeutics for theprophylaxis and treatment of venous thrombosis. Thrombosis Research,2012, 131:42-48. Westmuckett & Moore report a lack of tyrosylproteinsulfotransferase activity in hematopoietic cells drastically attenuatesatherosclerosis. Arterioscler Thromb Vasc Biol, 2009, 29:1730-1736.

In certain embodiments, the disclosure relates to methods of treating orpreventing a vascular disease or condition comprising administering aneffective amount of a pharmaceutical composition disclosed herein to asubject in need thereof.

In certain embodiments, the subject is a human that is at risk of,exhibiting symptoms of, or diagnosed with atherosclerosis, peripheralvascular disease, coronary heart disease, heart failure, rightventricular hypertrophy, cardiac dysrhythmia, endocarditis, inflammatorycardiomegaly, myocarditis, vascular heart disease, stroke,cerebrovascular disease, or peripheral arterial disease.

In certain embodiments, this disclosure relates to methods of treatingor preventing vascular disease or conditions such as atherosclerosis,atherosclerotic lesions, thrombus formation, thromboembolism, stroke, ormyocardial infarction by administering an effective amount of apharmaceutical composition disclosed herein to a subject in needthereof. In certain embodiments, the subject is at risk of, exhibitingsymptoms of, or diagnosed with atherosclerosis, atherosclerotic lesions,thrombus formation, thromboembolism, stroke, or myocardial infarction.

In certain embodiments, the subject has type I or type II diabetes,impaired glucose tolerance, elevated serum C-reactive proteinconcentration, vitamin B6 deficiency, dietary iodine deficiency,hypothyroidism, hyperlipidemia, hypertension, or is older than 50 yearsold, or smokes cigarettes daily.

In certain embodiments, the pharmaceutical composition is administeredin combination with a statin, atorvastatin, cerivastatin, fluvastatin,lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin,simvastatin, ezetimibe, amlodipine, niacin, aspirin, omega-3 fatty acid,or combinations thereof.

Selectin antagonists are also useful for the treatment of lung diseasessuch as asthma, bronchitis, emphysema, and COPD. Bimosiamose is apan-selectin antagonist that attenuates late asthmatic reactionsfollowing allergen challenge and attenuates airway inflammation in COPD.See Beeh et al., Pulm Pharmacol Ther, 2005, 19:233-4 and Watz et al.,Pulmonary Pharmacology & Therapeutics 26 (2013) 265-270. Schumacher etal. report that P-selectin glycopeptide ligand-1 (PSGL-1) isup-regulated on leukocytes from patients with chronic obstructivepulmonary disease. Clin Exp Immunol, 2005, 142:370-6.

In certain embodiments, the disclosure contemplates methods of treatingor preventing allergies or lung diseases or conditions comprisingadministering an effective amount of a pharmaceutical compositiondisclosed herein to a subject in need thereof. In certain embodiments,the subject is at risk of, exhibiting symptoms of, or diagnosed withasthma, bronchitis, emphysema, and COPD.

In certain embodiments, this disclosure related to using compounddisclosed herein for the treatment or prevention of inflammatorydisorders. In certain embodiments, the disclosure relates to thetreatment or prevention of inflammation or an inflammatory disordercomprising administering a compound disclosed herein to a subject inneed thereof. In certain embodiments, the inflammation is a result ofcardiac ischemia, injury, or a pathogenic infection, e.g. viral,bacterial, fungal, or the inflammatory disorder is selected fromatherosclerosis, allergies, acne vulgaris, asthma, autoimmune diseases,celiac disease, prostatitis, glomerulonephritis, hypersensitivities,inflammatory bowel disease, pelvic inflammatory disease, arthritis,rheumatoid arthritis, sarcoidosis, transplant rejection, vasculitis, orinterstitial cystitis.

In certain embodiments, the disclosure relates to treating inflammationor an inflammatory disease or condition by administering an effectiveamount of a compound disclosed herein in combination with ananti-inflammatory agent such as salicylates, aspirin (acetylsalicylicacid), diflunisal, salsalate, propionic acid derivatives, ibuprofen,dexibuprofen, naproxen, fenoprofen, ketoprofen, dexketoprofen,flurbiprofen, oxaprozin, loxoprofen, acetic acid derivatives,indomethacin, tolmetin, sulindac, etodolac, ketorolac, diclofenac,nabumetone, enolic acid (oxicam) derivatives, piroxicam, meloxicam,tenoxicam, droxicam, lornoxicam, isoxicam, fenamic acid derivatives(fenamates), mefenamic acid, meclofenamic acid, flufenamic acid,tolfenamic acid, selective COX-2 inhibitors (voxibs), celecoxib,rofecoxib, valdecoxib, parecoxib, lumiracoxib, etoricoxib, firocoxib,sulphonanilides, nimesulide, licofelone, and combinations thereof.

EXPERIMENTAL Compound Synthesis

Krishnamurthy et al. report the synthesis of an Fmoc-threonine bearingcore-2 glycan as a building block for PSGL-1 via Fmoc-assistedsolid-phase peptide synthesis. Carbohydr Res 2010, 345(11): 1541-1547.However, this method suffered from suboptimal regioselectivity in theglycosylation step (2.6:1). Furthermore, the approach required a triflicazide mediated diazotransfer, which due to the explosive nature of neatTfN₃. To circumvent these problems, our approach was revised to providea short, convenient route synthesis of glycoamino acid compound 7 withimproved regioselectivity and yield (See FIG. 2).

The synthesis began from 3,4,6 tri-O-acetyl-D-galactal, which could bereadily converted to a halide via a one pot azidochlorination step.Direct coupling of the chloride intermediate with a Fmoc-threonineacceptor was unsuccessful due to rapid decomposition. Therefore, thisintermediate was converted in situ to the thioglycoside donorcompound 1. Significantly, this two-step, one pot procedure could becarried out on a preparative scale (>50 gram) to provide donor compound1 in 67% yield. Coupling of compound 1 to the Fmoc-threonine acceptorproceeded to compound 2 in 78% yield with very high α-selectivity.De-O-acetylation of compound 2 under Zemplen conditions, selective 4,6acetal protection, and glycosylation with galactose donor compound 3under NIS/TfOH conditions, afforded benzylidene protected compound 4 in89% yield. Starting from 3,4,6 tri-O-acetyl-D-galactal, this diolcompound 5 is obtained in 7 steps in 21% overall yield.

In the glycosylation step with acceptor compound 5, the axial 4-OH groupwas anticipated to be of low reactivity, especially when carrying asubstituent at O-3. However, an undesired tetrasaccharide was identifiedin approximately 20% yield. Both desired and undesired compounds hadsimilar retention factor values. Thus, chromatographic separation waschallenging and laborious, particularly at a preparative scale. Toaddress this problem, low temperature activation (−10° C.) was performedwith glucosamine donor compound 6 at 0.8 Equiv (as opposed to 1.2Equiv), which produced only the β-glycoside compound 7 in 79% yield. The(1→6)-linkage in compound 7 was confirmed by NOESY spectrum, whichdisplayed a cross-peak between H-1 of the glucosamine residue and H-6 ofthe galactosamine residue. gHMBC NMR of compound 7 confirmed that O-6was glycosylated in revealing cross-peaks from 101.3 ppm (C-1 of theglucosamine residue, A-Cl) and 4.02 ppm (H-6 of the galactosamineresidue, BH6), as well as from 4.70 ppm (H-1 of the glucosamine residue,A-H1) and 69.5 ppm (C-6 of the galactosamine residue, B-C6). Acetylationof compound 7 with Py/Ac₂O, zinc reduction, and TFA mediated tert-butylester deprotection provided the Core-2 O-glycan compound 9 and offeredan alternative route to similar glycoamino acid building blocks forglycopeptide synthesis.

The glycopeptide binding site of PSGL-1 was synthesized using a Fmocassisted SPPS strategy (FIG. 2). Fmoc-Phe(CH₂SO₃H)—OH andFmoc-Phe(SO₃H)—OH amino acids were purchased from RSP Amino Acids LLC.Coupling reactions were performed using2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU) and 1-hydroxy-benzotriazol (HOBt). Fmoc groups were removed with20% piperidine/dimethylformamide without affecting the O-acetyl groupsor inducing β-elimination of the glycan. The presence of a N-fluorenyl(Fmoc) group allowed for photometric monitoring of the coupling reaction(λ=300.5 nm). For sulfonate mimics, Fmoc-Phe(CH₂SO₃)Na andFmoc-Phe(SO₃)Na coupling reactions proceeded smoothly, while couplingwith Fmoc-Tyr(OSO₃)Na amino acid was found to be difficult. The lattereffect was presumably due to the electron withdrawing nature of tyrosinesulfates, which deactivated the peptide sequence and reduced couplingefficiency. Deprotection was carried out using TFA mediated conditions(95% TFA/2.5% TES/2.5% H₂O) at room temperature for 1 h. The O-acetategroup was saponified using catalytic NaOMe in methanol to affordglycopeptide mimics GS_((n))P-3, which were further purified by RP-HPLC.When tyrosine O-sulfates were employed in peptide synthesis overallyield of GSP-3 was less than 0.5%. In contrast, the hydrolyticallystable sulfonate analogues resulted in a dramatic improvement of theoverall yield to afford GSnP-3 and GSn₂P-3 in 24% and 19% yields,respectively.

The sLe^(x) moiety was constructed on GS_((n))P-3 by usingglycosyltransferases in stepwise, sequential addition of galactose,sialic acid, and fucose. The highly efficient β1,4-galactosyltransferase(β1,4-GalT) is commercially available and was used to initially installβ1,4-galactose. Since sialyltransferases have low efficiency towards afucosylated Le^(x) structure, fucose was installed after sialylation.Thus, galactose was first appended in a β1,4 linkage to GlcNAc bytreatment with β1,4-GalT in the presence UDP-Galactose to produceGS(n)P-4. Similarly, addition of sialic acid was achieved by incubationof GsnP-4 with α2,3-sialyltransferase (α,2,3-(N)-sialylT) in thepresence of CMP-NeuAc to obtain GSnP-5. The sLe^(x) structure wascompleted by incubation of GS_((n))P-5 with humanα1,3-fucosyltransferase V (1,3-FucT V) and GDP-Fucose to produceGS_((n))P-6. Enzymatic steps typically proceeded with 60 to 70% yieldwithout observed interference of the sulfonate residues. In addition,desialylated glycopeptide mimics were generated through additionalsialylation of GSnP-4 to provide disialyl GSnP-6 (45% yield) followed byfucosylation to provide GsnP-7 in 55% yield. By utilizing this approach,four sets of glycopeptides mimics of PSGL-1 were synthesized, includingGSnP[3-6], disialyl GSnP-6, GSnP-7, GSn₂P[3-6], GSP[3-6], and three GSnPCore-1 peptides (FIG. 2).

Synthesis of Phenyl3,4,6-tri-O-acetyl-2-azido-2-deoxy-1-thio-β-D-galactoside 1

3,4,6-Tri-O-acetyl-D-galactal (50 g, 0.2 mol) was dissolved inacetonitrile (700 mL). Ferric chloride hexahydrate (40 g, 0.15 mol, 0.8eq.), sodium azide (13 g, 0.20 mol, 1.1 eq.) and hydrogen peroxide (21mL, 0.20 mol, 1.1 eq.; 33% aq. solution) were added subsequently and theclear red-brown solution was stirred at −20° C. for 1 h. After 1 h, 5 mLof hydrogen peroxide solution was further added and stirred foradditional 1 h at −10° C. The reaction mixture was diluted withdichloromethane (300 mL) and washed with water (4×70 mL), sat. NaHCO₃(2×100 mL), and NaCl solution (2×100 mL) until the organic layer wascolorless. ¹H-NMR (500 MHz, CDCl3): # [ppm]=2.07 (s, 3H), 2.08 (s, 3H),2.17 (s, 3H), 4.09-4.13 (m, 2H), 4.17 (dd, J=11.4, 3.4, 1H), 4.52 (m,1H), 5.38 (dd, J=10.8, 3.2, 1H), 5.51 (dd, J=3.1, 1.0, 1H), 6.18 (d,J=3.3 Hz, 1H); ¹³C-NMR (125.8 MHz, CDCl3): # [ppm]=20.6, 20.6, 20.7,58.5, 60.9, 66.8, 68.7, 69.7, 92.6, 169.6, 169.9, 170.5. The crudechloride intermediate (55 g, 0.15 mol) was subsequently dissolved indichlromethane (100 mL) and cooled to 0° C. To this solution was addedsodium hydride (7.2 g, 0.18 mol, 60% dispersion in mineral oil) andthiophenol (20 mL, 018 mol). The reaction mixture was stirred at to 0°C. for 1 h and slowly warmed up to room temperature. After 12 h, Thereaction mixture was diluted with dichlromethane (200 mL) and themixture was filtered through celite. The organic phase was separated,dried, and concentrated under reduced pressure. Subsequent purificationby chromatography over silica gel (Eluent: 40% ethyl acetate in hexane)thioglycoside donor 1 (α/β˜1/1) as colorless oil (52 g, 67% yield).1α-thioglycoside ¹H NMR (CDCl₃, 600 MHz) 1α: δ 1.99 (s, 3H, OAc), 2.09(s, 3H, OAc), 2.15 (s, 3H, OAc), 4.10 (2H, d, J 6.5 Hz, H-6), 4.33 (1H,dd, H-2), 4.78 (1H, t, H-5), 5.17 (1H, dd, J 11.1 Hz, H-3),), 5.19 (1H,dd, J 11.1 Hz, H-3), 5.51 (d, 1H, J 3.3 Hz, H-4), 5.72 (d, 1H, J 5.5 Hz,H-1), d 7.30-7.55 (m, 5H, aromatic). 1β-thioglycoside ¹H NMR (CDCl3): δ

δ 2.05 (s, 3H, OAc), 2.07 (s, 3H, OAc), 2.10 (s, 3H, OAc), 3.64 (1H, t,H-2), 3.91 (1H, t, H-5), 4.13 (1H, dd, J 6.3 Hz, H-6b), 4.19 (1H, dd, J6.8 Hz, 11.3 Hz, H-6a), 4.50 (1H, d, J 10.1 Hz, H-1), 4.88 (1H, dd, J10.3 Hz, H-3),), 5.40 (1H, d J 3.2 Hz, H-4), 7.32-7.65 (m, 5H,aromatic).

Synthesis ofN^(α)-(Fluoren-9-ylmethoxycarbonyl)-O—{O-(2′,3′,4′,6′-tetra-O-acetyl-β-D-galactopyranosyl)-(1→3)-O-[3″,4″,6″-tri-O-acetyl-2-deoxy-2-(2,2,2trichloroethoxycarbonylamino)-β-D-glucopyranosyl-(1→6)]-2-azido-2-deoxy-α-D-galactopyranosyl-L-threonineTert-butyl Ester 7:

Troc Glucosamine donor 6 (2.60 g, 4.8 mmol) and Core-1 acceptor 5 (2.80g, 3.2 mmol) were mixed with freshly activated 4 Å molecular sieves (2g). The reaction mixture was suspended in dichloromethane (50 mL) andcooled to −10° C. N-iodosuccinimide (1.8 g, 8.0 mmol) was slowly addedover the period of 30 min with vigorous suspension of the reactionmixture. Trifluoromethanesulfonic acid (700 μL, 0.8 mmol) was addedslowly and the stirring was continued at −10° C. for 2 h. The reactionmixture was filtered through celite into an aqueous solution of sodiumthiosulfate with disappearance of the dark red color as the solutionbecame colorless. The organic phase was separated, dried, andconcentrated under reduced pressure. Subsequent purification bychromatography over silica gel (Eluent: 60% ethyl acetate in hexane) toafford only the desired trisaccharide 7 (3.1 g, 79% yield). Analyticaldata for 7: [α]_(D) +45.4 (c 1, CHCl₃); ¹H NMR (CDCl₃, 600 MHz): δ 1.31(3H, d, J 6.0 Hz, Thr-CH₃), 1.51 (9H, s, -NHBoc), 1.98 (3H, s, OAc),2.00 (3H, s,OAc), 2.01 (3H, s, OAc), 2.02 (3H, s, OAc), 2.05 (3H, s,OAc), 2.08 (3H, s, OAc), 2.10 (3H, s, OAc), 3.54 (1H, d, J 10.8 Hz,Gal-N₃ H-2), 3.67 (1H, m), 3.74 (1H, m), 4.4-4.2 (12H, m), 4.6-4.5 (2H,m, Fmoc CH), 4.67 (1H, d, J 8.4 Hz, Thr CH^(β)), 4.70 (1H, d, J 12.0 Hz,GlcNTroc-1,6 H-1), 4.74 (1H, d, J 7.8 Hz, Gal H-1), 4.83 (1H, d, J 12.6Hz), 4.99 (1H, d, J 9.0 Hz, Gal N₃ H-1), 5.11 (3H, m), 5.26 (1H, ddd, J8.4 Hz, 12 Hz, 22.5 Hz, Gal H-2), 5.28 (1H, d, J 3.0 Hz, GlcNTroc-1,6H-3), 5.52 (1H, d, J 8.4 Hz), 5.61 (1H, d, J 9.6 Hz, Gal H-4), 6.07 (1H,d, J 9.6 Hz, Thr-NH), 7.16 (1H, d, J 6.6 Hz), 7.32 (2H, dd, J 7.2 Hz,7.8 Hz, -Fmoc Ar), 7.43 (2H, m, -Fmoc Ar), 7.63 (2H, d, J 7.2 Hz, -FmocAr), 7.77 (2H, d, J 7.8 Hz, -Fmoc Ar); ¹³C NMR (150 MHz, CDCl₃): 19.2,20.7, 20.8, 20.9, 20.9, 28.2, 47.3, 56.5, 58.7, 59.2, 61.5, 62.2, 67.0,67.7, 68.6, 68.7, 69.2, 69.3, 70.9, 71.3, 72.5, 72.1, 74.6, 75.6, 83.4,95.6, 99.2, 101.3, 102.2, 120.2, 125.1, 125.3, 127.2, 127.3, 127.9,141.5, 143.9, 144.0, 154.2, 157.1, 158.7, 159.3, 169.5, 169.7, 169.9,170.3, 170.4, 170.6, 170.8, 170.9; HRESIMS Calcd for C₅₈H₇₂O₂₇N₅Cl₃[M+K]⁺. 1414.31144; found 1414.31119.

Synthesis ofN^(α)-(Fluoren-9-ylmethoxycarbonyl)-O—{O-(2′,3′,4′,6″-tetra-O-acetyl-β-D-galactopyranosyl)-(1→3)-O-[3″,4″,6″-tri-O-acetyl-2-deoxy-2-acetamido-β-D-glucopyranosyl-(1→6)]-2-acetamido-2-deoxy-4-O-acetyl-α-D-galactopyranosyl-L-threonine9

The trisaccharide 9 was synthesized from 7 by the protocol inKrishnamurthy Carbohydr Res 2010, 345(11): 1541-1547 Analytical Data for9 [α]_(D) +35.7 (c 1, CHCl₃); ¹H NMR (CDCl₃, 600 MHz): δ_(H) 1.21 (3H,d, J 6.6 Hz, Thr-CH₃), 2.04 (30 H, s, 10×3 OAc's), 2.48 (1H, br. s,—OH), 3.43 (1H, t, J 8.4 Hz), 3.69-3.66 (2H, m), 3.83-3.79 (4H, m),4.13-4.04 (5H, m), 4.29-4.22 (5H, m), 4.42 (1H, d, J 6.0 Hz), 4.56-4.51(3H, m), 4.68 (1H, d, J 7.8 Hz), 4.84 (m, 1H), 4.92 (m, 1H), 5.03-4.97(m, 2H), 5.25 (m, 2H), 5.32 (1H, d, J 3.6 Hz), 7.30 (2H, ddd, J 3.0 Hz,7.8 Hz, 7.8 Hz, -Fmoc Ar), 7.39 (2H, ddd, J 4.2 Hz, 7.8 Hz, 7.8 Hz,-Fmoc Ar), 7.50 (1H, br. s —NH), 7.60 (2H, d, J 7.2 Hz, -Fmoc Ar), 7.76(2H, d, J 6.6 Hz, -Fmoc Ar); ¹³C NMR (150 MHz, CDCl₃): δ_(C) 18.5, 20.7,20.8, 20.9, 22.9, 23.1, 29.8, 47.4, 49.3, 49.4, 49.5, 49.7, 49.8, 50.0,54.8, 58.5, 61.1, 62.1, 66.7, 66.9, 68.5, 68.6, 68.7, 68.8, 69.5, 70.7,70.9, 72.0, 72.5, 76.1, 98.9, 99.0, 100.5, 101.0, 120.1, 125.0, 127.3,127.4, 127.9, 141.5, 143.9, 144.0, 157.0, 157.2, 169.7, 169.8, 170.6,170.8, 171.0, 171.1, 172.1; HRESIMS Calcd for C₅₇H₇₁O₂₈N₃ [M+H]⁺.1246.1769 found 1246.4297.

Procedure for Solid Phase Synthesis of Glycopeptides (GS_((n))P-3's):

The glycopeptides (GS_(n)P-3's) were synthesized manually on a NovasynTGA resin using standard Fmoc amino acid coupling strategy: Briefly,Fmoc-Leu-Novasyn TGA resin (5×4 μmol, 0.3 mmol/g) was loaded into apolypropylene centrifuge filter tube (0.22 μM micron, CorningInternational) equipped with a plastic cap. The resin in eachpolypropylene tube was swelled by stirring gently with 4×500 μLdichloromethane (DCM) for 10 min and filtered. The coupling reaction wasperformed with Fmoc-amino acids (24 μmol, 120 μL, 0.2 M), HBTU (24 μmol,120 μL, 0.2 M), HOBt (24 μmol, 120 μL, 0.3 M), DIPEA (36 μmol, 120 μL,0.3 M). The coupling steps were allowed for 2 h twice, except thesulfonate analogs whose coupling reaction was 10 h. Fmoc cleavage wasperformed with 20% piperidine in DMF (400 μL) for 10 min twice.Deprotection and side chain removal of glycopeptides was accomplished bygently stirring in 500 μL of TFA cocktail (95/2/2/1:TFA/H₂O/EDT/triethylsilane. (Note: For GS_((n))P-3 the cleavage step wasperformed at room temperature for 1 h. For GSP-3, the TFA cleavage stepneeded to be performed at 4° C. for 12 h as reported previously). Afterthis, the solution was evaporated and glycopeptides could beprecipitated by addition of 10 mL cold diethylether. Next, theglycopeptides was dissolved in methanol to which few drops of NaOMe wasadded, stirred for 1 h and subsequently purified by RP-HPLC (condition:water 75%-60%+0.1% TFA in 20 min for GS_((n))P-3 and water 80%-65%+100mM ammonium acetate in 20 minutes for GSP-3) and lyophilized to obtainGS_((n))P-3's as a white powder (GSnP-3 R_(t)=16.9 min; GSn₂P-3R_(t)=15.1 min; GSP-3 R_(t)=20.1 min). In MALDI-TOF analysis for GSnP-3the observed [M−H₂O]⁻ m/z is 3223.462 (calculated [M−H₂O]⁻C₁₄₁H₁₉₂N₂₀O₆₀S₃ m/z 3223.3261). For GSn₂P-3 the observed [M−H₂O]⁻ m/zis 3179.660 (calculated [M−H₂O]⁻ C₁₃₈H₁₈₆N₂₀O₆₀S₃m/z 3179.1280). ForGSP-3, the observed [M-3SO₃]⁻ for ammonium adduct is m/z 3021.742(calculated [M-3SO₃]⁻ C₁₃₈H₁₈₈N₂₁O₅₅ m/z 3021.0770).

Procedure for Synthesis of GS_((n))P-4:

GS_((n))P-3 (0.4 mM) was galactosylated using 125 milliunits of bovinemilk β1,4-GalT (Sigma) and UDP-Gal (1.5 mM) in a total volume of 9.5 mlof 40 mM sodium cacodylate, pH 7.0, 20 mM MnCl₂, and 0.02% NaN₃. After20 h of incubation at 37° C., a sample from the reaction mixture wasanalyzed by RP-HPLC which showed that all GS(n)P-3 had been convertedinto a faster moving product to afford GS_((n))P-4 (GSnP-4 R_(t)=16.0min; GSn₂P-4 R_(t)=14.9 min; GSP-4 R_(t)=19.5 min). Glycopeptide sampleswere deproteinated and desalted in a Sephadex G-50 column (10 ml,0.7-3.25 cm) using water or 25 mM NH₄HCO₃ as an eluant. 0.5-ml fractionswere collected, and the glycopeptides were detected by measuring UVabsorbance at 254 nm. In MALDI-TOF analysis for GSnP-4 the observed[M-10H]⁻ m/z is 3332.106 (calculated [M-10H]⁻ C₁₄₅H₁₉₂N₂₀O₆₄S₃ m/z3333.154): For GSn₂P-4. the observed [M-10H]⁻ for potassium adduct is3334.524 (calculated C₁₄₂H₁₈₇N₂₀O₆₄S₃ K m/z 3335.010): For GSP-4, theobserved [M-3SO₃]⁻ is m/z 3181.568 (calculated [M-3SO₃]⁻ C₁₄₅H₁₉₈N₂₀O₆₀m/z 3181.2216).

Procedure for Synthesis of GS_((n))P-5:

GS_((n))P-4 (1 mM) was sialylated using 20 milliunits ofα2,3-(N)-sialylT (Calbiochem, La Jolla, Calif.) and 3 mM CMP-NeuAc(Sigma) in a total volume of 6.0 ml of 50 mM MOPS, pH 7.4, 0.1% bovineserum albumin, and 0.02% NaN₃. After 14 h of incubation at 37° C., thesample was analyzed by RP-HPLC, which showed that GS(n)P-4 had beenconverted completely into a faster moving product, GS(n)P-5 (GSnP-5R_(t)=15.8 min; GSn₂P-5 R_(t)=14.3 min; GSP-5 R_(t)=19.1 min). InMALDI-TOF analysis for GSnP-5 the observed [M-NH₃]⁻ m/z is 3621.655(calculated C₁₅₆H₂₁₈N₂₀O₇₂S₃m/z 3621.685): For GSn₂P-5. the observed[M-10H]⁻ for potassium adduct is 3625.112 (calculated C₁₅₃H₂₀₄N₂₁O₇₂S₃K[M-10H]⁻ m/z 3624.647). For GSP-5, the observed [M-3SO₃]⁻ is m/z3471.994 (calculated [M-3SO₃]⁻ C₁₅₆H₂₁₅N₂₁O₆₈ m/z 3472.4762).Disialylation of GSnP-4 provided Disialyl-GSnP-6 compound in 45% yield:In MALDI-TOF analysis for Disialyl-GSnP-6 the observed [M-10H]⁻ m/z is3915.048 (calculated [M-10H]⁻ C₁₆₇H₂₂₆N₂₂O₈₀S₃ m/z 3915.345).

Procedure for Synthesis of GS_((n))P-6

3-FucT-VI-GS(n)P-5 (0.4 mM) was α1,3-fucosylated for 16 h at 37° C. with2 milliunits of α1,3-Fucosyltransferase-VI (Calbiochem) and GDP-Fucose(0.8 mM) (Calbiochem) in a total volume of 3.5 ml of 50 mM MOPS, pH 7.4,20 mM MnCl₂ and 0.02% NaN₃. Deproteinated and desalted sample wasanalyzed by RP-HPLC, which showed that GS(n)P-5 was converted completelyinto the product GS_((n))P-6 (GSnP-6 R_(t)=15.5 min; GSn₂P-6 R_(t)=14.1min; GSP-6 R_(t)=19.0 min). Starting with 12 mg of GSnP-3, the overallrecovery of GSnP-6 was 5.5 mg, as determined by UV absorbance at 275 nmduring the HPLC runs. In MALDI-TOF analysis for GSnP-6 the observed[M-NH₃]⁻ m/z is 3766.923 (calculated [M-NH₃]⁻ C₁₆₂H₂₂₇N₂₀O₇₆S₃ m/z3766.819): In ESI-QTOF analysis for GSnP-6 the observed triply chargedspecies [M−H]³⁻ m/z is 1259.7906 (calculated [M−H]³⁻ C₁₆₂H₂₂₈N₂₁O₇₆S₃m/z 1259.7927); Observed doubly charged species [M−H]²⁻ m/z is 1890.1857(calculated [M-2H]²⁻ C₁₆₂H₂₂₈N₂₁O₇₆S₃ m/z 1890.916): In MALDI-TOFanalysis for GSn₂P-6, the observed [M-3SO₂]⁻ is 3769.587 (calculatedC₁₆₁H₂₂₉N₂₁O₇₆S₃ [M-CO₂]⁻ m/z 3768.38621). For GSP-6, the observed[M-3SO₃]⁻ is m/z 3615.470 (calculated [M-3SO₃]⁻ C₁₆₂H₂₂₅N₂₁O₇₂ m/z3615.4590). Fucosylation of Disialyl-GSnP-6 compound afforded GSnP-7 in55% yield. In MALDI-TOF analysis for GSnP-7 the observed m/z [M-10H]⁻4061.042 (calculated [M-10H]⁻ C₁₇₃H₂₃₆N₂₂O₈₄S₃ m/z 4061.40338.

GSnP-6 Demonstrates Nanomolar Affinity to P-Selectin.

Binding affinities of PSGL-1 mimics towards selectins was initiallyscreened using a microarray in which glycosulfopeptides and glycanstandards (Sialyl Le^(x), NA2, NA2,3, NA2,6 and LNnT) were printed on aNHS-activated glass slide. The slide was then incubated with recombinantIg chimeras of P-, L-, or E-selectin (5-20 μg/mL) followed by Alexa-488labeled anti-human IgG antibody (5 μg/mL). Similar to a native PSGL-1sequence containing tyrosine sulfate (GSP-6), GSnP-6 bound to P-selectinmore strongly than to E- or L-selectin. Likewise, GSnP-7, a sialylatedextension of GSnP-6, showed higher affinity to P-selectin. However,disialyl GSnP-6, lacking the fucosyl residue displayed lower affinity toP-selectin consistent with the key contribution of α-1,3-fucose. Bindingof glycopeptides mimics to selectins was Ca²⁺ dependent and inhibited byEDTA.

Dissociation constants (K_(d)) were determined using a Biacore bindingassay after initial capture of biotinylated GS_((n))P-6 ontostreptavidin-coated sensor chips followed by flow through of P-, L- orE-sel-Ig (2.5 to 60 μg/mL). Dissociation constants for GSnP-6 andGSn₂P-6 to human P-selectin were 22 nM and 14 nM, respectively; comparedto the K_(d) of 73 nM for native PSGL-1. The K_(d) of GSnP-6 to murineP-selectin was ˜9-fold lower than to human P-selectin. GSnP-6 bound toE- and L-selectin with even lower apparent affinity.

The chemical stability of GSnP-6 was evaluated under low pH conditionsby incubating the compound in sodium acetate/acetic acid buffer at pH 5,37° C., as well as under high temperature conditions by incubation in 2mM phosphate buffer at pH 7.5, 60° C. (FIG. 3E). The native N-terminalPSGL-1 sequence, GSP-6 rapidly degrades under these conditions, whereasdegraded products were not detected for GSnP-6 by RP-HPLC.

GSnP-6 Inhibits P-Selectin/PSGL-1 Dependent Interactions In Vitro.

Flow cytometry was used to characterize the ability of GSnP-6 to blockbinding of selectin-IgG chimeras to murine and human leukocytes.Recombinant mouse P- or L-selectin Fc chimera (2 μg/mL) was incubatedwith murine neutrophils and GSnP-6 (0-30 μM). Likewise, recombinanthuman P-selectin or L-selectin Fc chimera were incubated with humanperipheral blood neutrophils and monocytes, as well as the human U937cell line in the presence of GSnP-6 (0-30 μM). Binding of selectin-IgGchimeras was detected with PE-conjugated anti-IgG antibody, quantifiedas mean fluorescent intensity, and plotted as percent inhibition.Specificity was confirmed with selectin-specific blocking antibodies.GSnP-6 inhibited P-selectin dependent interactions in a dose dependentmanner in all four cell lines, including human U-937 cells (IC₅₀˜8 μM),murine PMN (IC₅₀˜15 μM), human monocytes (IC₅₀˜30 μM), human PMN(IC₅₀˜30 μM. GSnP-6 also inhibited PSGL-1/L-selectin interactions, asassessed with human U-937 cells (IC₅₀˜30 μM) and murine neutrophils(IC₅₀>50 μM).

GSnP-6 Inhibits Leukocyte Rolling In Vivo.

Intravital microscopy was used to determine the ability of GsnP-6 toinhibit leukocyte binding to microvascular endothelium. PSGL-1 expressedon circulating leukocytes binds to selectins on endothelium; inducingleukocyte rolling to facilitate subsequent tight binding to integrinreceptors. Inhibition of this interaction leads to an increase inrolling velocity. Intravital microscopy of the murine cremaster musclemicrocirculation was performed on venules with a diameter of 30 to 40μm. Seven venules per mouse (n=4, saline (control); n=3, GSnP-6, 4gmol/kg IV) were analyzed and the velocities of 5 to 10 rollingleukocytes were determined in each venule by individually trackingleukocyte distance/time (μm/s). A significant increase in mean rollingvelocity was observed after intravenous administration of GSnP-6 (Fig.GsnP-6: 74.9±3.4 μm/s, saline: 40.8±1.7 μm/s; p≤0.01).

1-22. (canceled)
 23. A method of treating cancer in a subject comprisingadministering to the subject an effective amount of a glycopeptidecomprising Y¹X¹Y²X²X³Y³X⁴X⁵X⁶Z¹X⁷W¹ (SEQ ID NO: 1), or a salt thereof,wherein: W¹ is threonine or serine substituted with a saccharide orpolysaccharide; X¹, X², X³, X⁴, X⁵, X⁶, and X⁷ are individually andindependently any amino acid; Y¹, Y², and Y³ are each individually andindependently tyrosine, phenylalanine, or phenylglycine unsubstituted orsubstituted with —SO₃H, —CH₂SO₃H, —CF₂SO₃H, —CO₂H, —CONH₂, —NHSO₂CH₃,—SO₂NH₂, or CH₂PO₃H; and Z¹ is proline or hydroxyproline.
 24. The methodof claim 23, wherein at least one or two of Y¹, Y², and Y³ aresubstituted with —CH₂SO₃H.
 25. The method of claim 23, wherein Y¹, Y²,and Y³ are substituted with —CH₂SO₃H.
 26. The method of claim 23,wherein the polysaccharide is sialyl Lewis X or sialyl Lewis A.
 27. Themethod of claim 23, wherein the polysaccharide comprises2-(acetylamino)-2-deoxy-galactose alpha 1 bonded to W¹, galactose beta 3bonded to 2-(acetylamino)-2-deoxy-galactose,2-(acetylamino)-2-deoxy-glucose beta 6 bonded to2-(acetylamino)-2-deoxy-galactose, and fucose alpha 3 bonded to2-(acetylamino)-2-deoxy-glucose.
 28. The method of claim 23, wherein thepolysaccharide comprises 2-(acetylamino)-2-deoxy-galactose alpha 1bonded to W¹, galactose beta 3 bonded to2-(acetylamino)-2-deoxy-galactose, 2-(acetylamino)-2-deoxy-glucose beta6 bonded to 2-(acetylamino)-2-deoxy-galactose, fucose is alpha 3 bondedto 2-(acetylamino)-2-deoxy-glucose, and galactose beta 4 bonded to2-(acetylamino)-2-deoxy-glucose.
 29. The method of claim 23, wherein thepolysaccharide comprises 2-(acetylamino)-2-deoxy-galactose alpha 1bonded to W¹, galactose beta 3 bonded to2-(acetylamino)-2-deoxy-galactose, 2-(acetylamino)-2-deoxy-glucose beta6 bonded to 2-(acetylamino)-2-deoxy-galactose, fucose is alpha 3 bondedto 2-(acetylamino)-2-deoxy-glucose, galactose beta 4 bonded to2-(acetylamino)-2-deoxy-glucose, and5-acetamido-3,5-dideoxy-glycero-galacto-2-nonulosonic acid alpha 3bonded to galactose.
 30. The method of claim 23, wherein thepolysaccharide comprises 2-(acetylamino)-2-deoxy-galactose alpha 1bonded to W¹, galactose beta 3 bonded to2-(acetylamino)-2-deoxy-galactose, 2-(acetylamino)-2-deoxy-glucose beta6 bonded to 2-(acetylamino)-2-deoxy-galactose, galactose beta 4 bondedto 2-(acetylamino)-2-deoxy-glucose,5-acetamido-3,5-dideoxy-glycero-galacto-2-nonulosonic acid alpha 3bonded to galactose, and fucose alpha 3 bonded to2-(acetylamino)-2-deoxy-glucose.
 31. The method of claim 23, wherein thepolysaccharide comprises 2-(acetylamino)-2-deoxy-galactose alpha 1bonded to W¹, a first galactose beta 3 bonded to2-(acetylamino)-2-deoxy-galactose, 2-(acetylamino)-2-deoxy-glucose beta6 bonded to 2-(acetylamino)-2-deoxy-galactose, a second galactose beta 4bonded to 2-(acetylamino)-2-deoxy-glucose, fucose is alpha 3 bonded to2-(acetylamino)-2-deoxy-glucose, and5-acetamido-3,5-dideoxy-glycero-galacto-2-nonulosonic acid alpha 3bonded to the first galactose.
 32. The method of claim 23, wherein X¹,X³, X⁴, and X⁷ is each individually and independently E, D, N, or Q. 33.The method of claim 23, wherein X², X⁵, and X⁶ is each individually andindependently L, I, V, A or F.
 34. The method of claim 23, wherein theglycopeptide comprises (SEQ ID NO: 2) Y¹EY²LDY³DFLZ¹EW¹E, (SEQ ID NO: 3)Y¹EY²LDY³DFLZ¹EW¹EP, (SEQ ID NO: 4) Y¹EY²LDY³DFLZ¹EW¹EPL, (SEQ ID NO: 5)EY¹EY²LDY³DFLZ¹EW¹, (SEQ ID NO: 6) EY¹EY²LDY³DFLZ¹EW¹E, (SEQ ID NO: 7)EY¹EY²LDY³DFLZ¹EW¹EP, (SEQ ID NO: 8) EY¹EY²LDY³DFLZ¹EW¹EPL,(SEQ ID NO: 9) KEY¹EY²LDY³DFLZ¹EW¹, (SEQ ID NO: 10)KEY¹EY²LDY³DFLZ¹EW¹E, (SEQ ID NO: 11) KEY¹EY²LDY³DFLZ¹EW¹EP, or(SEQ ID NO: 12) KEY¹EY²LDY³DFLZ¹EW¹EPL.


35. The method of claim 23, wherein the glycopeptide comprises(SEQ ID NO: 12) KEY¹EY²LDY³DFLZ¹EW¹EPL.


36. The method of claim 35, wherein W¹ is threonine.
 37. The method ofclaim 35, wherein Z¹ is proline.
 38. The method of claim 23, wherein theglycopeptide comprises KEY¹EY²LDY³DFLZ¹EW¹EPL (SEQ ID NO: 12), or saltthereof, wherein: W¹ is threonine; Y¹, Y², and Y³ are phenylalanine4-substituted with —CH₂SO₃H; Z¹ is proline;2-(acetylamino)-2-deoxy-galactose alpha 1 bonded to W¹, galactose isbeta 3 bonded to 2-(acetylamino)-2-deoxy-galactose,2-(acetylamino)-2-deoxy-glucose is beta 6 bonded to2-(acetylamino)-2-deoxy-galactose, fucose is alpha 3 bonded to2-(acetylamino)-2-deoxy-glucose, Galactose is beta 4 bonded to2-(acetylamino)-2-deoxy-glucose, and5-acetamido-3,5-dideoxy-glycero-galacto-2-nonulosonic acid alpha 3bonded to galactose.
 39. The method of claim 23, wherein theglycopeptide comprises (SEQ ID NO: 4) Y¹EY²LDY³DFLZ¹EW¹EPL,(SEQ ID NO: 8) EY¹EY²LDY³DFLZ¹EW¹EPL, (SEQ ID NO: 9)KEY¹EY²LDY³DFLZ¹EW¹, (SEQ ID NO: 10) KEY¹EY²LDY³DFLZ¹EW¹E,(SEQ ID NO: 11) KEY¹EY²LDY³DFLZ¹EW¹EP, or (SEQ ID NO: 12)KEY¹EY²LDY³DFLZ¹EW¹EPL.


40. The method of claim 23, wherein the cancer is a hematologicalmalignancy, a leukemia, lymphoma, acute lymphoblastic leukemia (ALL),acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL),small lymphocytic lymphoma (SLL), chronic myelogenous leukemia, acutemonocytic leukemia (AMOL), Hodgkin's lymphomas, non-Hodgkin's lymphomas,Burkitt lymphoma, B-cell lymphoma, multiple myelomacervical, ovariancancer, colon cancer, breast cancer, gastric cancer, lung cancer,melanoma, skin cancer, ovarian cancer, pancreatic cancer, prostatecancer, head cancer, neck cancer, or renal cancer.
 41. The method ofclaim 23, for preventing tumor metastasis in the subject.
 42. The methodof claim 23, wherein the subject is a human.